CN111349637B - Mycoplasma caprae and subspecies pneumoniae specific proteins and indirect ELISA kit - Google Patents
Mycoplasma caprae and subspecies pneumoniae specific proteins and indirect ELISA kit Download PDFInfo
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Abstract
The invention discloses a goat mycoplasma goat subspecies pneumonia specific protein 87.1 and an indirect ELISA kit, wherein the protein 87.1 has a nucleic acid sequence shown as SEQ ID NO.1, and the indirect ELISA antibody detection kit prepared by the goat mycoplasma goat subspecies pneumonia specific protein 87.1 comprises an ELISA plate coated by antigen protein 87.1, sample diluent, positive serum, negative serum, washing liquid, blocking liquid, rabbit anti-sheep secondary antibodies, substrate chromogenic liquid and stop liquid. The coating amount of the antigen protein 87.1 was 625 ng/well, and the dilutions of positive serum and negative serum were 1/100. The indirect ELISA kit prepared from the Mccp specific protein 87.1 provided by the invention has good specificity, reacts with Mccp positive serum, and does not react with Mmc and Mcc positive serum.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mycoplasma caprae pneumonitis subspecies specific protein and an indirect ELISA kit.
Background
Goat infectious pleuropneumonia (contagious caprine pleuropneumonia, CCPP) is a serious respiratory disease in goats caused by Mycoplasma capricolum subsp. Mcp belongs to the mycoplasma filamentous cluster, the members of which have a high degree of serological and genomic similarity, and in addition to mcp, also include: mycoplasma filiform subspecies (M.mycides subsp. Mycides, mmm), mycoplasma caprae subspecies (M.mycides subsp. Capri, mmc), mycoplasma caprae subspecies (M.capricolumsubsp. Capricolum, mcc), mycoplasma Li Jishi (M.Leachii, ml), wherein Mmm and Ml are Mycoplasma bovis, mmc, mcc and Mccp are Mycoplasma capricolum, each causing capricopneumonia. Closest to Mccp relatedness are Mcc and Ml, which are susceptible to cross-reactivity with Mccp in serological assays. The key to determining the specificity and sensitivity of serological diagnostic methods is antigen, mainly by mining mcp-specific antigen in two directions, on the one hand, with the rapid development of genomic sequencing technology, the complete genomic sequence of 10 mcp strains has been resolved. 26 potential virulence factors have been reported and shown to be recognized by the host immune system as potential serological diagnostic targets. On the other hand, from protein level, by IgG immunoblotting test, it was found that the experimental infected serum and 25 parts of field serum all appeared to be striped at 44, 40 and 23Kd, and 108, 70 and 62Kd stripes were observed in most of the serum; and 6 candidate diagnosis proteins of the mycoplasma caprae subspecies are obtained by a 2D electrophoresis mass spectrometry and western blot comparison proteomics method, and comprise a pyruvate dehydrogenase complex, a glycine phosphate kinase, a pyridine-nucleoside phosphatase, a 30S ribosomal protein S6, triethyl diphosphate and an amide acetyl transferase. These are all available as candidate targets for Mccp-specific antigens, but no report has been made as to whether these candidate antigens could be further used.
Disclosure of Invention
Aiming at the defects pointed out in the background technology, the invention provides a goat mycoplasma goat subspecies pneumoniae specific protein and an indirect ELISA kit, which can react with Mccp positive serum and do not react with Mmc and Mcc positive serum, and have good specificity.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a mycoplasma caprae and subspecies caprae pneumonitis specific protein has an expression sequence of a protein 87.1, wherein the protein 87.1 has a nucleic acid sequence shown as SEQ ID NO. 1.
The invention further provides application of the mycoplasma caprae pneumonitis subspecies specific protein in preparation of an indirect ELISA antibody detection kit for mycoplasma caprae pneumonitis subspecies.
The invention further provides an indirect ELISA antibody detection kit prepared from the goat mycoplasma goat subspecies pneumonitis specific protein, which comprises an ELISA plate coated with the antigen protein 87.1, sample diluent, positive serum, negative serum, washing liquid, blocking liquid, rabbit anti-goat secondary antibody, substrate chromogenic liquid and stop liquid.
Preferably, the coating amount of the antigen protein 87.1 is 625 ng/hole, and the dilution of the positive serum and the negative serum is 1/100.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
the invention provides a mcp specific protein 87.1, which is characterized in that the mcp specific sequence 87.1 is obtained through bioinformatics analysis, then the recombinant protein 87.1 is obtained through a prokaryotic expression technology, and the specificity and reactivity of the 87.1 are initially evaluated by establishing an indirect ELISA kit, so that the 87.1 reacts with mcp positive serum, can reflect the change of the antibody level, and does not react with Mmc and Mcc positive serum.
Drawings
FIG. 1 is an expression form of a mycoplasma caprae pneumonitis subspecies specific protein 87.1 provided by an embodiment of the present invention; in the figure, M: protein Marker,1-3 is 87.1 bacterial liquid, wherein 1: supernatant after bacterial liquid ultrasound, 2: precipitation after sonication, 3: un-sonicated bacterial fluid; 4: pET-30a no-load control.
FIG. 2 is a graph showing the purification effect of the Mycoplasma caprae pneumonias subspecies specific protein 87.1 inclusion body provided by the example of the present invention. In the figure, C:8M urea lysate PH6.3, 1/2/3 th time of impurity washing liquid; d:8M urea lysate PH5.9, 1/2/3 th time of impurity washing liquid; e:8M urea lysate pH4.5, 1/2/3/4 th eluate.
FIG. 3 shows the results of evaluation of the reactivity of the mycoplasma caprae pneumonitis subspecies specific proteins provided by the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
1. Experimental materials
The main reagent comprises: plasmid pET-30a, competent BL 21 (DE 3), LB, LA, IPTG, QIGEN resin, skimmed milk powder, secondary antibody (rabbit anti-goat, sigma), mccp antibody detection cELISA kit (IDEXX Co.), BCA kit, urea.
The main instrument is as follows: shaking table, incubator, ultrasonic breaker, centrifuge, protein electrophoresis apparatus, enzyme-labeled instrument. Serum: were taken at different time points in vaccine/infection experiments.
2. Sequence acquisition
The sequence of the mcp 1601 genome (nz_cp 017125.1) was BLAST-compared to the sequences of the other members Mmc, mcc, mmm, ml of the mycoplasma pool to obtain a partial mcp-specific sequence, which was aligned in-line at NCBI and found to be all present in the sequenced 10 mcp genomic sequence.
3. Sequence analysis screening
The molecular weight is used for preliminary screening, and the structural functions of the sequences are predicted by on-line prediction software, including antigen index, surface accessibility, hydrophilicity index, cell positioning, transmembrane region and signal peptide. The sequence with high antigen index, good hydrophilicity, high surface accessibility, no signal peptide and less transmembrane region is selected to obtain the sequence of the recombinant protein more easily by a prokaryotic expression technology, and finally the 87.1 (NZ_CP 017125.1, complexe (740269.. 740802)) sequence is determined as an expression sequence, wherein the 87.1 has a nucleic acid sequence shown as SEQ ID NO. 1.
4. Construction of pET-30a-87.1 plasmid
In mycoplasma, the codon TGA is not used as a stop codon, but is translated into tryptophan, so that TGA codon in the sequence is replaced by TGG, then the sequence is optimized by a prokaryotic expression codon optimizing system, the N, C end is respectively inserted into BamHI and XhoI enzyme cutting sites, the N end is inserted into His tag, the sequence is synthesized by a company, pET-30a is selected as an expression vector, the expression vector pET-30a-87.1 is constructed, and enzyme cutting and sequencing verification are carried out.
5. 87.1 prokaryotic expression of protein
The constructed pET-30a-87.1 plasmid is transferred into competent BL21 (DE 3) for expression, a single colony on a susceptance plate is selected to be inoculated into LB for induction expression in the same specification, conditions of different induction temperatures (16/26/37 ℃), induction times (4/8/12/16 h) and IPTG concentration (0.1/0.5/1 mM) are searched, the result shows that under all conditions, 87.1 expression forms are mainly inclusion bodies, the size is about 26Kd, a small amount of supernatant is expressed but the purification effect is poor, according to the expression quantity, finally, 37 ℃ and 4h and 1mM IPTG are selected as induction conditions, elution is carried out with different PH eluents, protein electrophoresis verification is carried out, and BCA quantification is carried out. Finally, the inclusion body protein 87.1 was successfully purified, and the result is shown in FIG. 2.
6. Indirect ELISA System set-up
By optimizing the dilution of the antigen antibody and the blocking solution conditions, the iELISA reaction conditions were: antigen 625 ng/well, 100 μl per well, diluted with 0.05M PH9.6 carbonate coating buffer, incubated for 1h at 37deg.C in incubator, and then overnight at 4deg.C; washing the plate 3 times by 0.01M PBST, and sealing the plate for 2 hours by a sealing liquid in a 37 ℃ incubator; plates were washed 3 times with 0.01M PBST, positive serum was also 100-fold diluted with sample dilution, 100 μl per well, incubated for 1h at 37 ℃; washing the plate 4 times with 0.01M PBST, diluting the secondary antibody with 5000 times of sample diluent, and incubating for 1h at 37 ℃ with 100 μl of each well; plates were washed 4 times with 0.01M PBST, developed by adding 100. Mu.l TMB to each well, and incubated at 37℃for 10min;100 μl 2M H 2 SO 4 Terminating the reaction per well; reading OD on a microplate reader 450 nm Values.
7. Specificity and reactogenicity assessment
Serum from different time points during vaccine experiments is taken for mcp antibody detection, and serum from 8 sheep at 5 different time points comprises immunization for 0 day, immunization for 3 weeks, and challenge for 0 day/immunization for 1 month, and challenge for 2 weeks, and challenge for 3 weeks, totaling 40 parts. In addition, because mycoplasma ovis Mcc and Mmc and Mccp are easy to cross react in serological detection method, 2 parts of Mmc and Mcc positive serum are detected, and the specificity is estimated to be 87.1.
The result of the reactivity evaluation is shown in fig. 3, and through preliminary verification, the Mccp antibody can be detected by 87.1, and the change of the antibody level at different time points can be reflected, so that the antibody has the reactivity; and did not react with Mmc, mcc positive serum, as shown in Table 1, again demonstrating the specificity of the 87.1 protein.
TABLE 1 87.1 specificity evaluation
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Sequence listing
<110> the animal doctor institute of Lanzhou, china academy of agricultural sciences
<120> goat mycoplasma goat subspecies pneumoniae-specific protein 87-1 and indirect ELISA kit
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 534
<212> DNA
<213> Mycoplasma pneumoniae
<400> 1
ttattttcaa gaactttttt tcatatcact gtcatttaca acaccatatt catctacaaa 60
taaaatatca cattctaatc ctttagtgtt tatatatttt gtgattgtca tgaattttga 120
atttaaagaa tcaattcttt ttttcaaatt gttcacagaa gaatttgtgt gtgataaaaa 180
aattttactt ttatcattaa atatttgcga catataattt attaaagttg tttttcctga 240
acctgcagaa ccatatataa atgcaacttg tgtatctcta aatatttttt gaattataat 300
tttcttttct tctgaagaaa tattattctt attaagtcac ttatctgaca gattactata 360
accttgataa cctttttttg tgtatgtttt cagtttttct attataaaat ctaagttgtt 420
agcatattta tgaatatata agtaatttat ttcattatcg ggtctatgac caaatcacaa 480
tttttgatta taagataaaa ttattttttt caattttacc tgtataatca acat 534
Claims (3)
1. The application of mycoplasma caprae pneumonitis subspecies specific protein in preparing an indirect ELISA antibody detection kit for mycoplasma caprae pneumonitis subspecies is characterized in that the expression sequence of the specific protein is a protein 87.1 sequence, and the protein 87.1 is encoded by a gene with a nucleic acid sequence shown as SEQ ID NO. 1.
2. An indirect ELISA antibody detection kit prepared from mycoplasma caprae pneumonitis subspecies specific protein is characterized by comprising an ELISA plate coated with antigen protein 87.1, sample diluent, positive serum, negative serum, washing liquid, blocking liquid, rabbit anti-goat secondary antibody, substrate chromogenic liquid and stop liquid, wherein the protein 87.1 is encoded by a gene with a nucleic acid sequence shown as SEQ ID NO. 1.
3. The indirect ELISA antibody detection kit of claim 2, wherein the antigen protein 87.1 is coated at 625 ng/well and the dilutions of positive and negative serum are 1/100.
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Citations (3)
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| WO2017011919A1 (en) * | 2015-07-22 | 2017-01-26 | University Of Saskatchewan | Mycoplasma vaccines and uses thereof |
| CN110229919A (en) * | 2019-06-26 | 2019-09-13 | 宁夏大学 | For detecting the composition, kit and method of Mycoplasma bovis |
| CN110596402A (en) * | 2019-09-12 | 2019-12-20 | 广西壮族自治区兽医研究所 | ELISA kit for rapidly detecting mycoplasma ovipneumoniae antibody |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017011919A1 (en) * | 2015-07-22 | 2017-01-26 | University Of Saskatchewan | Mycoplasma vaccines and uses thereof |
| CN110229919A (en) * | 2019-06-26 | 2019-09-13 | 宁夏大学 | For detecting the composition, kit and method of Mycoplasma bovis |
| CN110596402A (en) * | 2019-09-12 | 2019-12-20 | 广西壮族自治区兽医研究所 | ELISA kit for rapidly detecting mycoplasma ovipneumoniae antibody |
Non-Patent Citations (4)
| Title |
|---|
| Alonso JMM 等."Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp mycoides SC".《VETERINARY MICROBIOLOGY》.2002,第89卷(第4期),277-289. * |
| Chu Y. 等."Mycoplasma capricolum subsp. capripneumoniae M1601 chromosome, complete genome".《GenBank》.2019,Accession：NZ_CP017125.1. * |
| 吴娅琴."虹鳟源鲁氏耶尔森菌主要生物学特性及减毒活疫苗研究".《中国优秀硕士学位论文全文数据库 农业科技辑》.2021,(第1期),D050-574. * |
| 王慧 等."绵羊肺炎支原体DnaK B细胞抗原表位预测及其蛋白结构分析".《生物技术通报》.2014,第260卷(第3期),159-164. * |
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